Using Alternating Electric Fields to Increase Permeability of the Blood Brain Barrier

ABSTRACT

Certain substances (e.g., large molecules) that ordinarily cannot traverse the blood brain barrier can be introduced into the brain by applying an alternating electric field to the brain for a period of time, wherein the frequency of the alternating electric field is selected so that application of the alternating electric field increases permeability of the blood brain barrier. In some embodiments, the frequency of the alternating electric field is less than 190 kHz (e.g., 100 kHz). Once the permeability of the blood brain barrier has been increased, the substance is able to cross the blood brain barrier.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application62/722,100, filed Aug. 23, 2018, which is incorporated herein byreference in its entirety.

BACKGROUND

Ordinarily, cerebral microvessels strictly regulate the transfer ofsubstances between the blood and the brain tissue. This regulation bycerebral micro-vessels is called the blood-brain barrier (BBB), and isdue to intercellular tight junctions (TJs) that form between braincapillary endothelial cells. In cerebral capillaries, TJs proteins areexpressed 50-100 times more than in peripheral microvessels. TJs areformed by an intricate complex of transmembrane proteins (claudin andoccludin) with cytoplasmic accessory proteins (ZO-1 and -2, cingulin,AF-6, and 7H6). By linking to the actin cytoskeleton, these proteinsform a strong cell-cell connection. Brain endothelial cells, which formthe endothelium of cerebral microvessels, are responsible for about75-80% of the BBB's resistance to substances, and other cells such asastrocytes and pericytes provide the remainder of the resistance.

The BBB consists of tight junctions around the capillaries, and itordinarily restricts diffusion of microscopic objects and large orhydrophilic molecules into the brain, while allowing for the diffusionof hydrophobic molecules (transcellular instead of paracellulartransport).

In healthy people, the BBB serves a very important function because itprevents harmful substances (e.g. bacteria, viruses, and potentiallyharmful large or hydrophilic molecules) from entering the brain. Thereare, however, situations where the action of the BBB introducesdifficulties. For example, it might be desirable to deliver large orhydrophilic drug molecules to treat a disease in the patient's brain.But when the BBB is operating normally, these drugs are blocked fromentering the brain by the BBB.

SUMMARY OF THE INVENTION

One aspect of the invention is directed to a first method for deliveringa substance across a blood brain barrier of a subject's brain. In thisfirst method, the relevant substance can be delivered across a bloodbrain barrier of a subject's brain by applying an alternating electricfield to the subject's brain for a period of time. Application of thealternating electric field to the subject's brain for the period of timeincreases permeability of the blood brain barrier in the subject'sbrain. The substance is administered to the subject after the period oftime has elapsed, and the increased permeability of the blood brainbarrier allows the substance to cross the blood brain barrier.

In some instances of the first method, the alternating electric field isapplied at a frequency between 75 kHz and 125 kHz. In some instances ofthe first method, the period of time is at least 24 hours. In someinstances of the first method, the period of time is at least 48 hours.In some instances of the first method, the alternating electric fieldhas a field strength of at least 1 V/cm in at least a portion of thesubject's brain. In some instances of the first method, the alternatingelectric field is applied at a frequency between 75 kHz and 125 kHz, theperiod of time is at least 24 hours, and the alternating electric fieldhas a field strength of at least 1 V/cm in at least a portion of thesubject's brain.

In some instances of the first method, the administering of thesubstance is performed intravenously. In some instances of the firstmethod, the administering of the substance is performed orally. In someinstances of the first method, the subject's brain is tumor-free.

In some instances of the first method, the substance comprises a drugfor treating a disease. Examples of these instances include a cancertreatment drug, an infectious disease treatment drug, aneurodegenerative disease treatment drug, or an auto-immune diseasetreatment drug, an anti-epileptic drug, a hydrocephalus drug, a strokeintervention drug, or a psychiatric drug. In some instances of the firstmethod, the substance is used for monitoring brain activity. Examples ofthese instances include a brain dye, a reporter, or a marker.

In any of the instances of the first method noted above, discontinuingthe application of the alternating electric field may be done to allowthe blood brain barrier to recover.

Another aspect of the invention is directed to a second method fordelivering a substance across a blood brain barrier of a subject'sbrain. In this second method, the relevant substance can be deliveredacross a blood brain barrier of a subject's brain by applying analternating electric field at a first frequency to the subject's brainfor a period of time, wherein the first frequency is less than 190 kHzand the period of time is at least 24 hours, wherein application of thealternating electric field at the first frequency to the subject's brainfor the period of time increases permeability of the blood brain barrierin the subject's brain. The substance is administered to the subjectafter the period of time has elapsed, and the increased permeability ofthe blood brain barrier allows the substance to cross the blood brainbarrier.

In some instances of the second method, the alternating electric fieldis applied at a frequency between 75 kHz and 125 kHz. In some instancesof the second method, the period of time is at least 48 hours. In someinstances of the second method, the alternating electric field has afield strength of at least 1 V/cm in at least a portion of the subject'sbrain. In some instances of the second method, the alternating electricfield is applied at a frequency between 75 kHz and 125 kHz, and thealternating electric field has a field strength of at least 1 V/cm in atleast a portion of the subject's brain.

In any of the instances of the second method noted above, discontinuingthe application of the alternating electric field may be done to allowthe blood brain barrier to recover.

The methods described herein may be used to deliver a substance acrossthe blood brain barrier of a subject's whose brain is tumor free. Inthis situation, another aspect of the invention is directed to a thirdmethod for delivering a substance across a blood brain barrier of asubject's brain. In this third method, the relevant substance can bedelivered across a blood brain barrier of a subject's brain that doesnot include a tumor by applying an alternating electric field at a firstfrequency to the subject's brain for a period of time. The applicationof the alternating electric field at the first frequency to thesubject's brain for the period of time increases permeability of theblood brain barrier in the subject's brain. The substance isadministered to the subject after the period of time has elapsed, andthe increased permeability of the blood brain barrier allows thesubstance to cross the blood brain barrier.

In some instances of the third method, the alternating electric field isapplied at a frequency between 75 kHz and 125 kHz. In some instances ofthe third method, the period of time is at least 24 hours. In someinstances of the third method, the period of time is at least 48 hours.In some instances of the third method, the alternating electric fieldhas a field strength of at least 1 V/cm in at least a portion of thesubject's brain. In some instances of the third method, the alternatingelectric field is applied at a frequency between 75 kHz and 125 kHz, theperiod of time is at least 24 hours, and the alternating electric fieldhas a field strength of at least 1 V/cm in at least a portion of thesubject's brain.

In any of the instances of the third method noted above, discontinuingthe application of the alternating electric field may be done to allowthe blood brain barrier to recover.

The methods described herein may be used to deliver a substance acrossthe blood brain barrier of a subject with a brain tumor. In thissituation, another aspect of the invention is directed to a fourthmethod for treating a tumor in a subject's brain and delivering asubstance across a blood brain barrier of the subject's brain. In thisfourth method, a first alternating electric field is applied at a firstfrequency to the subject's brain for a first period of time. Applicationof the first alternating electric field at the first frequency to thesubject's brain for the first period of time increases permeability ofthe blood brain barrier in the subject's brain. The substance isadministered to the subject after the first period of time has elapsed,and the increased permeability of the blood brain barrier allows thesubstance to cross the blood brain barrier. A second alternatingelectric field at a second frequency is applied to the subject's brainfor a second period of time that is at least one week long. The secondfrequency is different from the first frequency, and the secondalternating electric field at the second frequency has an intensity thatis sufficiently large to inhibit the tumor.

In some instances of the fourth method, the first frequency is between75 kHz and 125 kHz.

In some instances of the fourth method, the first frequency is between50 kHz and 190 kHz. In some of these instances, the second frequency isbetween 190 kHz and 210 kHz.

In some instances of the fourth method, the first period of time is atleast 24 hours. In some instances of the fourth method, the secondperiod of time comprises a single uninterrupted interval of time that isat least one week long. In other instances of the fourth method, thesecond period of time comprises a plurality of non-contiguous intervalsof time during which the second alternating electric field at the secondfrequency is applied to the subject's brain, wherein the plurality ofnon-contiguous intervals of time collectively add up to at least oneweek.

In any of the instances of the fourth method noted above, discontinuingthe application of the alternating electric field may be done to allowthe blood brain barrier to recover.

In some instances, any of the methods described above is used to delivera substance having a molecular weight of at least 4 kDa across a bloodbrain barrier of a subject's brain.

In some instances, any of the methods described above is used to delivera substance having a molecular weight of at least 69 kDa across a bloodbrain barrier of a subject's brain.

In some instances, any of the methods described above is used to delivera substance across a blood brain barrier of a subject's brain, whereinthe substance has at least one characteristic that ordinarily impedesthe substance from crossing a non-leaky BBB.

Another aspect of the invention is directed to a first apparatus fortreating a tumor in a subject's body and facilitating delivery of asubstance across a blood brain barrier of the subject's body. The firstapparatus comprises an AC voltage generator capable of operating at afirst frequency between 50 and 190 kHz and a second frequency between 50and 500 kHz. The second frequency is different from the first frequency.The AC voltage generator has a control input, and the AC voltagegenerator is configured to output the first frequency when the controlinput is in a first state and to output the second frequency when thecontrol input is in a second state. The first apparatus also comprises acontroller programmed to (a) place the control input in the second stateso that the AC voltage generator outputs the second frequency, (b)accept a request to switch to the first frequency, (c) upon receipt ofthe request, place the control input in the first state so that the ACvoltage generator outputs the first frequency for an interval of time,and (d) after the interval of time has elapsed, place the control inputin the second state so that the AC voltage generator outputs the secondfrequency.

Some embodiments of the first apparatus further comprise a set ofelectrodes configured for affixation to the subject's body; and wiringthat connects an output of the AC voltage generator to the set ofelectrodes.

In some embodiments of the first apparatus, the first frequency isbetween 75 kHz and 125 kHz, and the second frequency is between 150 kHzand 250 kHz. In some embodiments of the first apparatus, the interval oftime is at least 24 hours. In some embodiments of the first apparatus,the interval of time is at least 72 hours. In some embodiments of thefirst apparatus, the controller is further programmed to, subsequent tothe receipt of the request, switch the control input back and forthbetween the first state and the second state.

In some embodiments of the first apparatus, the AC voltage generator iscapable of operating at at least one additional frequency between 50 and500 kHz, and the AC voltage generator is configured to output the atleast one additional frequency when the control input is in at least oneadditional state, and the controller is programmed to cycle the controlinput through the second state and the at least one additional stateprior to receipt of the request, and to cycle the control input throughthe second state and the at least one additional state after theinterval of time has elapsed.

Some embodiments of the first apparatus further comprise a userinterface, and the request is accepted via the user interface. In someembodiments of the first apparatus, the request is accepted via radiofrequency (RF).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary setup for in vitro experiments in whichimortilized murine brain capillary endothelial cells (cerebEND) weregrown on coverslips and transwell inserts to create an artificial invitro version of the BBB.

FIGS. 2A and 2B depict the results of integrity and permeabilitytesting, respectively, on the artificial BBB.

FIGS. 3A and 3B depict data showing that the increased permeability ofthe artificial BBB is not caused by cell death.

FIG. 4 depicts the positions at which rats' brains were sliced for an invivo experiment.

FIG. 5 depicts the EB accumulation for this in vivo experiment indifferent sections of the rats' brains.

FIG. 6 depicts the average EB accumulation in the rat brain for this invivo experiment, averaged over all sections.

FIG. 7 depicts the increase in BBB permeability in three differentsections of rat brains that is induced by alternating electric fields invivo, as determined using contrast enhancement MRIs.

FIG. 8 depicts the increase in BBB permeability in rat cortexes that isinduced by alternating electric fields in vivo, as determined usingcontrast enhancement MRIs.

FIG. 9 depicts a suitable timing relationship between the application ofthe alternating electric field and the administration of the substanceto the subject.

FIG. 10 is a block diagram of a dual-frequency apparatus that generatesa first frequency for inducing BBB permeability and a second frequencyfor inducing cytotoxicity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This application describes a novel approach for temporarily increasingthe permeability of the BBB using alternating electric fields so thatsubstances that are ordinarily blocked by the BBB will be able to crossthe BBB.

A set of in vitro experiments was run in which imortilized murine braincapillary endothelial cells (cerebEND) were grown on coverslips andtranswell inserts to create an artificial in vitro version of the BBB,and FIG. 1 depicts the setup for these experiments. The cells were thentreated with alternating electric fields (100-300 kHz) for 24 h, 48 h,and 72 h. The direction of the alternating electric fields was switchedevery 1 second between two perpendicular directions (i.e., 1 second inone direction followed by 1 second in the other direction, in arepeating sequence). The following effects were then analyzed: (a) Cellmorphology (immunofluorescence staining of tight junction proteinsClaudin 5 and ZO-1); (b) BBB integrity (using transendothelialelectrical resistance (TEER)); and (c) BBB permeability (usingfluoresceine isothiocyanate coupled to dextran (FITC) for flowcytometry).

A first set of experiments involved visualization of cell morphology andorientation, and visualization of the localization of stained proteins.This experiment was designed to ascertain how the frequency of thealternating electric field impacted the artificial BBB. Here, the cellswere grown on coverslips, and alternating electric fields were appliedfor 72 hours at four different frequencies (100 kHz, 150 kHz, 200 kHz,and 300 kHz), with a field strength of 1.7 V/cm. The direction of thealternating electric fields was switched every 1 second between twoperpendicular directions. There was also a control in which alternatingelectric fields were not applied. Cell morphology images depicting thepresence of Claudin 5, ZO-1, and 4,6-diamidino-2-phenylindole (DAPI) in(each of which was stained a different color) were then obtained.Claudin 5 and ZO-1 indicate the presence of an intact BBB. This set ofcell morphology images revealed that alternating electric fields disturbthe artificial BBB by delocalization of tight junction proteins from thecell boundaries to the cytoplasm, with the most dramatic effects at 100kHz.

A second set of experiments also involved visualization of cellmorphology. This experiment was designed to ascertain how the durationof time during which the alternating electric field was applied impactedthe artificial BBB. Endothelial cells were grown on coverslips, and analternating electric field at a frequency of 100 kHz was applied forthree different durations (24 h, 48 h, 72 h) plus a control. Thedirection of the alternating electric fields was switched every 1 secondbetween two perpendicular directions. Cell morphology images depictingthe presence of Claudin 5 and DAPI (each of which was stained adifferent color) were then obtained. This set of cell morphology imagesrevealed that the phenomena discussed above in connection with the firstset of experiments were already visible after 24 hours, and that theeffects were most pronounced after 72 hours.

A third set of experiments also involved visualization of cellmorphology. This experiment was similar to the second set ofexperiments, except that the endothelial cells were grown on transwellinserts instead of coverslips. The results were similar to the resultsof the second set of experiments. The delocalization of TJ proteins wasvisible after 24 hours and the effects were most pronounced after 72hours. The three experiments described above support the conclusion thatalternating electric fields cause structural changes in the cells, whichmight be responsible for an increase in BBB permeability.

FIGS. 2A and 2B depict the results of integrity and permeabilitytesting, respectively, on the artificial BBB after subjecting it toalternating electric fields at a frequency of 100 kHz for 72 hours (withthe direction of the alternating electric fields switched every 1 secondbetween two perpendicular directions), and for a control. Morespecifically, FIG. 2A depicts the results of a transendothelialelectrical resistance (TEER) test, which reveals that alternatingelectric fields reduced the integrity of the artificial BBB to 35% ofthe control. FIG. 2B depicts the results of a fluoresceineisothiocyanate (FITC) permeability test, which reveals that thealternating electric fields increased the permeability of the artificialBBB to FITC-dextrans with a 4 kDa molecular weight to 110% of thecontrol. These experiments further support the conclusion thatalternating electric fields increase the permeability of the BBB tomolecules that ordinarily cannot traverse a non-leaky BBB.

Collectively, these in vitro experiments reveal that applyingalternating electric fields at certain frequencies for a sufficientduration of time causes the delocalization of tight junction proteins(Claudin 5, ZO-1) from the cell boundaries to the cytoplasm (with themost dramatic effects at 100 kHz), and increases the permeability of theBBB. The alternating electric fields' effects appear already after 24 hand are most prominent after 72 h. More specifically, after using thealternating electric fields to increase the permeability of the BBB,molecules of 4 kDa can pass through the BBB.

Additional in vitro experiments were then conducted to determine whathappens to the BBB after the alternating electric fields were turnedoff. These experiments used visualization of cell morphology to show howthe artificial BBB recovers after discontinuing the alternating electricfields. In these experiments, endothelial cells were grown on coverslipsand treated with 100 kHz alternating electric fields at a field strengthof 1.7 V/cm for 72 hours. The direction of the alternating electricfields was switched every 1 second between two perpendicular directions.The alternating electric fields were then turned off, and the cells werefollowed for 96 hours after stopping the alternating electric field.Cell morphology images depicting the presence of Claudin 5 (stained)were obtained at 24 hours, 48 hours, 72 hours, and 96 hours. Thoseimages revealed a progressive change in localization of Claudin betweenthe cell boundaries and the cytoplasm on the 24 h, 48 h, 72 h, and 96 himages. Furthermore, a comparison of those four images to the respectiveimages for the control (in which alternating electric fields were notapplied during either the first 72 h or the last 96 h) revealed that theendothelial cell morphology was partially recovered 48 hours afterstopping the alternating electric fields, and that the BBB was fullyrecovered (i.e., was comparable to the control) 96 hours after stoppingthe alternating electric fields.

FIGS. 3A and 3B depict the results of an in vitro experiment designed todetermine whether the observed changes in the permeability of theartificial BBB described above might be attributable to cell death. Thisexperiment tested cell division by comparing cell counts (a) whenalternating electric fields were applied for 72 hours followed by noalternating electric fields for 96 hours with (b) a control in whichalternating electric fields were never applied. Endothelial cells weregrown on coverslips and treated with 100 kHz alternating electric fieldsat a field strength of 1.7 V/cm for 72 hours. The direction of thealternating electric fields was switched every 1 second between twoperpendicular directions. The alternating electric fields were thenturned off, and the cells were followed for 96 hours after stopping thealternating electric field. The number of cells per ml for thealternating electric fields and the control were counted, and theresults are depicted in FIGS. 3A and 3B (for control and for alternatingelectric fields, respectively). These results reveal that there was nostatistically significant increase in the cell numbers during or afterapplication of the alternating electric fields, which indicates that thechanges in the BBB permeability noted above could not be attributed tocell death.

Another in vitro experiment used a TUNEL assay for apoptosis todetermine whether the observed changes in the permeability of theartificial BBB described above might be attributable to cell death. Inthis experiment, endothelial cells were grown on coverslips and treatedwith 100 kHz alternating electric fields at a field strength of 1.7 V/cmfor 72 hours. The direction of the alternating electric fields wasswitched every 1 second between two perpendicular directions. In thecontrol, alternating electric fields were not applied. Cell morphologyimages depicting apoptosis (TUNEL) and Nuclei (DAPI) (each of which wasstained a different color) were obtained after 24, 48, and 72 hours.None of those images revealed additional evidence of apoptosis,indicating that alternating electric fields did not cause cell death.This confirms that the changes in the BBB permeability noted above werenot attributable to cell death.

A set of in vivo experiments on rats was also run to quantify theincrease in vessel permeability caused by exposure to the alternatingelectric fields. These experiments used Evans Blue (EB) dye, which is anazo dye that has a very high affinity for serum albumin (molecule size˜69 kDa). Because of its large molecule size, serum albumin willordinarily not be able to get past the BBB. But if the permeability ofthe BBB has been sufficiently increased, some of the serum albuminmolecules (together with the EB dye that has been bound thereto) willmake it across the BBB and can then be detected by looking for the EB inthe rat's brain.

In this set of experiments, 100 kHz alternating electric fields wereapplied to the rat's brain for 72 hours, and the direction of thealternating electric fields was switched every 1 second between twoperpendicular directions. This was accomplished by shaving each rat'shead, positioning a first pair of capacitively coupled electrodes on thetop and bottom of the rat's head, and positioning a second pair ofcapacitively coupled electrodes on the left and right sides of the rat'shead. A 100 kHz AC voltage was then applied between the top and bottomelectrodes for 1 second, followed by a 100 kHz AC voltage appliedbetween the right and left electrodes for 1 second, in a repeatingsequence.

Under the conditions indicated in Table 1 and for the times indicated onTable 1, EB was injected intravenously into the tail vein underanesthesia (Once injected, EB immediately binds to Albumin), and the EBwas allowed to circulate for 2 hours in all cases. The following stepswere then performed: (a) intracardiac perfusion with saline; (b) brainsare sliced in four pieces with a brain slicer; (c) pieces werephotographed to localize staining and weighted; (d) EB extraction aftertissue homogenization with TCA 50% (1:3) and centrifuge and (e) EBquantification at 610 nm. Results are given as μg EB per g tissue.

TABLE 1 Group # of # Treatment Rats Time of EB Injection 1 72 hours 100kHz fields 3 2 h before the end of the 72 h period 2 72 hours 100 kHzfields + 3 2 h after the end of the 2 hours rest 72 h period 3 72 hoursheat using sham 3 2 h before the end of heat electrodes 4 Control (nofields + no heat) 3 Same time as group 1

During the experiment, two animals from group 2 and one animal fromgroup 4 were excluded (disrupted treatment, failure to inject EB intothe tail vein). There were no differences between the animals treatedwith alternating electric fields (groups 1 and 2) and therefore theseanimals were grouped together. Similarly, there were no differencesbetween sham heat and control animals (groups 3 and 4) and thereforethese animals were grouped together.

The rats' brains were sliced into four pieces using a brain slicer atthe positions shown in FIG. 4. EB accumulation in these four specificsections was then measured. In addition, a computer simulation wasperformed to determine the field strength in each of these foursections. Table 2 specifies the field strength obtained from thesimulation in each of these four sections, with all values given in V/cmRMS.

TABLE 2 Section 1 2 3 4 Mean Field Strength 2.7 V/cm 3 V/cm 2.6 V/cm 1.6V/cm Media Field Strength 2.5 V/cm 2.6 V/cm 2.4 V/cm 1.6 V/cm

The results for EB accumulation in sections 1 through 4 are depicted inFIG. 5. The summary of these results is as follows: (1) a statisticallysignificant increase was observed in sections 1, 2 (frontal cerebrum)where the field strength was highest; and a smaller increase (that wasnot statistically significant) was observed in the more posteriorsections (3, 4) where the field strength was lower.

FIG. 6 depicts the average EB accumulation in the rat brain, averagedover all four sections 1-4. This result reveals higher accumulation ofEB in the brains of rats treated with alternating electric fields for 72hours, and this result was statistically significant (p<0.05).

The in vivo experiments described above establish that: (1) alternatingelectric fields application permits the BBB passage of molecules ofaverage molecular size of ˜69 kDa to the brain tissue; (2) the increasein permeability of the BBB is maintained 2 hours after terminating thealternating electric fields application; and (3) the increasedpermeability of the BBB varies between different sections of the brain.The latter may be the result of the different field strengths that wereimposed in the various sections of the brain. These experiments furthersupport our conclusion that alternating electric fields increase thepermeability of the BBB to molecules that ordinarily cannot traverse anon-leaky BBB.

In another set of in vivo experiments, 5 rats were treated withalternating electric fields at 100 kHz for 72 h, and 4 control rats werenot treated with alternating electric fields for the same period oftime. At the end of the 72 hour period, the fluorescent compoundTRITC-Dextran of 4 kDa was injected intravenously into the tail veinunder anesthesia, and allowed to circulate for 2 minutes in all cases.The brains were then removed, frozen, sectioned and scanned with afluorescent scanner. All slides were scanned with the same conditions.The resulting images revealed significantly higher levels ofaccumulation of the fluorescent 4 kDA TRITC-Dextran in the brain tissueof the rats that were subjected to alternating electric fields (ascompared to the control), confirming yet again that alternating electricfields increase the permeability of the BBB.

Yet another set of in vivo experiments was performed using DynamicContrast Enhanced MRI (DCE-MRI) with intravenous injection of Gadoliniumcontrast agent (Gd-DTPA, Magnetol, MW 547). In these experiments, testrats were treated with 100 kHz alternating electric fields for 72 h, andcontrol rats were not treated with alternating electric fields for thesame period of time. After this 72 h period, the alternating electricfield was turned off, the rats were anesthetized, and a series of 60 T1wMRI scans (each of the scans having a duration of 28 seconds) wasacquired. The gadolinium contrast agent was injected into the rat's tailvein during the 7th of these 60 scans.

The image analysis for each rat included (1) determining a baseline foreach voxel by calculating the mean of the first six T1w MRI scans foreach voxel (i.e., the scans prior to the injection of the gadolinium);(2) computing voxel by voxel, the percent signal change (i.e.,gadolinium accumulation) over time relative to the baseline; (3)segmenting the brain into anterior, middle, and posterior segments; (4)generating for each of the three segments the mean percent signal changewith respect to the baseline over all the voxels in the respectivesegment and then (5) averaging 4 consecutive time points (i.e. 4 scans)together. Finally, the data from all of the rats within any given groupwere averaged together.

The results of this DCE-MRI experiment for each of the three segments ofthe brain (i.e. anterior, middle, and posterior) are depicted in FIG. 7.This data reveals that contrast agent accumulation in the brain tissueof rats that were treated with alternating electric fields (tracelabeled TTFields; n=6) was significantly higher than in the control rats(trace labeled control; n=3). Moreover, the distinction was mostpronounced in the posterior brain, which is the portion of the brainwhere the alternating electric fields had the highest field strength.From this we can conclude that the alternating electric fieldssuccessfully increased the permeability of the BBB in vivo.

To test whether this increase in permeability of the BBB was temporary,the same test conditions were repeated, but followed with an additional96 hours without alternating electric fields. After this 96 hour period,a series of 60 T1w MRI scans (each of the scans having a duration of 28seconds) was acquired using the same procedure described above(including the gadolinium injection). The results of this portion of theDCE-MRI experiment for each of the three segments of the brain are alsodepicted in FIG. 8. This data reveals that contrast agent accumulationin the brain tissue of rats that were treated with alternating electricfields for 72 hours followed by 96 hours without alternating electricfields (trace labeled TTFields+96 h; n=7) was not significantlydifferent from the control rats (trace labeled control+96 h; n=3). Fromthis we can conclude that the permeability of the BBB returns to normalafter the alternating electric fields are discontinued.

An additional series of 60 T1w MRI scans (each of the scans having aduration of 28 seconds) was also acquired using the same procedurebefore the alternating electric field was applied to the rats (n=2). Theresults of this portion of the DCE-MRI experiment for each of the threesegments of the brain (i.e. anterior, middle, and posterior) are alsodepicted in FIG. 8 (see the trace labeled “before”).

FIG. 8 shows the average of all 3 segments of the brain (i.e. anterior,middle, and posterior) for 72 h of TTFields (n=6) and a control of noTTFields for 72 h (n=3), with standard deviation bars. A paired t testwas used to compare between the two groups, and p<0.0001.

We note that the upper size limit of molecules that can pass through theBBB after applying the alternating electric fields has not yet beendetermined. But based on (a) the in vitro experiments described hereinusing FITC-dextrans with a 4 kDa molecular weight and (b) the in vivoexperiments described herein using EB (which binds to serum albuminhaving a molecule size of ˜69 kDa), the upper limit appears to be atleast about 69 kDa, and is most certainly at least 4 kDa.

The implications of being able to reversibly increase the permeabilityof the BBB at will are far-reaching, because it now becomes possible todeliver many substances across the BBB of a subject, despite the factthat those substances have at least one characteristic that ordinarilyimpedes the substance from crossing a non-leaky BBB. Many of theseimplications involve delivering a substance including but not limited totreating agents and diagnostic agents across a blood brain barrier of asubject's brain.

Examples include but are not limited to the following: deliveringchemotherapeutic agents across the BBB to treat cancer (in this context,it may be possible to lower the dosage of drugs for the treatment ofbrain tumors and metastases with severe side effects in other parts ofthe body based on the increased permeability of the drugs to the brain);delivering antibodies and/or cell-based therapies across the BBB forimmunotherapy; delivering contrast agents dyes, reporters, and markersacross the BBB for diagnostic purposes and for research (e.g. monitoringbrain activity); delivering antibacterial agents across the BBB to treatinfectious diseases; delivering anti-viral agents or virus neutralizingantibodies across the BBB to treat viral infections; deliveringanti-parasitic agents across the BBB to treat parasites; deliveringagents to treat neurodegenerative and autoimmune disease across the BBB;delivering psychiatric drugs; delivering anti-epileptic drugs;delivering hydrocephalus drugs; delivering stroke intervention andrecovery drugs; delivering compounds that are lacking in the brainacross the BBB to treat conditions in which those compounds are lacking(e.g., for treating Parkinson's disease, etc.).

While the testing described above was done in vitro and in live rats, itis expected that similar results will be obtained with other animals andwith humans.

The methods described herein can also be applied in the in vivo contextby applying the alternating electric fields to a live subject's brain.Imposing the electric field in the subject's brain will increase thepermeability of the BBB, which will enable molecules that are ordinarilyblocked or impeded by the BBB to get through. This may be accomplished,for example, by positioning electrodes on or below the subject's skin sothat application of an AC voltage between selected subsets of thoseelectrodes will impose the alternating electric fields in the subject'sbrain.

For example, one pair of electrodes could be positioned on the front andback of the subject's head, and a second pair of electrodes could bepositioned on the right and left sides of the subject's head. In someembodiments, the electrodes are capacitively coupled to the subject'sbody (e.g., by using electrodes that include a conductive plate and alsohave a dielectric layer disposed between the conductive plate and thesubject's body). But in alternative embodiments, the dielectric layermay be omitted, in which case the conductive plates would make directcontact with the subject's body. In another embodiment, electrodes couldbe inserted subcutaneously below a patent's skin.

An AC voltage generator applies an AC voltage at a selected frequency(e.g., 100 kHz, or between 50 and 190 kHz) between the right and leftelectrodes for a first period of time (e.g. 1 second), which inducesalternating electric fields where the most significant components of thefield lines are parallel to the transverse axis of the subject's head.Then, the AC voltage generator applies an AC voltage at the samefrequency (or a different frequency) between the front and backelectrodes for a second period of time (e.g. 1 second), which inducesalternating electric fields where the most significant components of thefield lines are parallel to the sagittal axis of the subject's head.This two step sequence is then repeated for the duration of thetreatment. Optionally, thermal sensors may be included at theelectrodes, and the AC voltage generator can be configured to decreasethe amplitude of the AC voltages that are applied to the electrodes ifthe sensed temperature at the electrodes gets too high. In someembodiments, one or more additional pairs of electrodes may be added andincluded in the sequence. In alternative embodiments, only a single pairof electrodes is used, in which case the direction of the field lines isnot switched. Note that any of the parameters for this in vivoembodiment (e.g., frequency, field strength, duration,direction-switching rate, and the placement of the electrodes) may bevaried as described above in connection with the in the vitroembodiments. But care must be taken in the in vivo context to ensurethat the electric field remains safe for the subject at all times.

A wide variety of applications for increasing the permeability of theBBB can be readily envisioned in the in vivo context. In one example,localized enhancement of drug uptake by tumor cells (e.g., glioblastomacells) in the brain can be induced by applying alternating electricfields to the brain for a period of time (e.g., 72 hours or at least 24hours) prior to and during administration of chemotherapies or otherantineoplastic agents.

FIG. 9 depicts a suitable relationship in timing between the applicationof the alternating electric field and the administration of thesubstance to a live patient. Based on the data described above, andassuming that the substance is introduced or administered at a giventime t=0, the alternating electric field can begin before the given time(e.g., 72 hours before t=0), and continue for an interval of time afterthe given time (e.g., until 12 hours following t=0). In this situation,the permeability of the BBB will begin to increase before the substanceis administered and before the substance reaches the BBB. This willenable the substance to cross the BBB immediately upon its arrival. Inthe context of chemotherapy, this would correspond to startingapplication of the alternating electric fields, administering thechemotherapeutic agent 72 hours later, followed by application of thealternating electric fields for an additional interval of time (e.g.,until 12 hours following the time at which the chemotherapeutic agentwas administered).

Note that the intervals of time discussed above in connection with FIG.9 can either be uninterrupted or can include breaks that are preferablyshort. For example, a 12 hour interval could be satisfied by a singleuninterrupted block of 12 hours. Alternatively, the 12 hour intervalcould be satisfied by applying the alternating electric fields for 6hours, followed by a 1 hour break, followed by applying the alternatingelectric fields for an additional 6 hours. Similar breaks may alsooptionally interrupt the 72 hour interval that precedes theadministration of the substance. Note also that in the context of FIG.9, when the substance is administered to a live patient, theadministration of the substance may be performed using any of a varietyof approaches including but not limited to intravenously, orally,subcutaneously, intrathecal, intraventricularly, and intraperitonealy.

In some preferred embodiments, the frequency of the alternating electricfields is less than 190 kHz (e.g., between 50 and 190 kHz or between 25and 190 kHz). Based on the experiments discussed above, using afrequency of less than 190 kHz combined with a period of time of atleast 24 hours will increase the change in permeability (as compared tooperating outside of those ranges).

The methods described herein may be used to deliver a substance acrossthe blood brain barrier of a subject's brain when the subject's brainincludes a tumor. One existing approach to treating brain tumors (e.g.,glioblastoma) is by applying alternating electric fields at frequenciesbetween 50 and 500 kHz, preferably between 100 and 300 kHz to the tumor.For glioblastoma, 200 kHz is the most preferred frequency. Alternatingelectric fields at these frequencies are referred to as TTFields, andare described in U.S. Pat. Nos. 6,868,289 and 7,565,205, each of whichis incorporated herein by reference in its entirety. Briefly, those twoapplications describe disrupting dividing cells during mitosis. Theeffectiveness of TTFields is improved when the direction of the electricfield is periodically switched, when the strength of the field in atleast a portion of the tumor is at least 1 V/cm, and when the fields areapplied for long periods of time (e.g., weeks or months) with as fewbreaks as possible.

In patients with brain tumors, situations may arise where it will bedesirable to treat the tumor with TTFields and also deliver a substanceacross the same patient's blood brain barrier (e.g., to help get atherapeutically effective amount of a chemotherapy drug past the BBB toprovide an additional line of attack against the tumor). In somesituations, it may be possible to use a single frequency of analternating electric field to both treat the tumor and increase thepermeability of the BBB. In other situations, it may be desirable to usealternating electric fields with different frequencies: a firstfrequency that is selected to provide improved results for increasingthe permeability of the BBB, and a second frequency that is selected toprovide improved results for the anti-tumor action of the TTFields.

FIG. 10 is a block diagram of an apparatus that generates a firstfrequency for inducing BBB permeability and a second frequency forinducing cytotoxicity. The apparatus includes an AC voltage generator 44that is similar to the conventional Optune® field generator unit, buthas the ability to operate at two different frequencies. The firstfrequency is between 50 and 190 kHz and the second frequency is between50 and 500 kHz. In some embodiments, the first frequency is between 75kHz and 125 kHz and the second frequency is between 150 kHz and 250 kHz.

The ability to operate at two different frequencies may be implemented,for example, using relays to switch either a first set of components ora second set of components into the conventional circuit that generatesthe AC voltage, and adjusting the operating frequency of an oscillator.The AC voltage generator 44 is configured to output either the firstfrequency or the second frequency depending on the state of a controlinput. When the control input is in a first state the AC voltagegenerator 44 outputs the first frequency, and when the control input isin a second state the AC voltage generator 44 outputs the secondfrequency. A controller 42 is programmed to place the control input inthe second state so that the AC voltage generator 44 outputs the secondfrequency. The controller 42 is also programmed to accept a request toswitch to the first frequency. In the embodiment depicted in FIG. 10,the request arrives via a user interface 40 that may be implementedusing any of a variety of conventional approaches including but notlimited to a pushbutton, a touch screen, etc. In alternativeembodiments, the request may arrive via RF (e.g. Bluetooth, WiFi, etc.)from a tablet, smartphone, etc.

Upon receipt of the request, the controller 42 will place the controlinput in the first state so that the AC voltage generator 44 will outputthe first frequency for an interval of time (e.g., 72 hours). After theinterval of time has elapsed, the controller 42 will place the controlinput in the second state so that the AC voltage generator 44 reverts tooutputting the second frequency.

Optionally, the AC voltage generator 44 may be configured to output oneor more additional frequencies (e.g., a third frequency, a fourthfrequency, etc.), depending on the state of the control input.Preferably each of these additional frequencies is selected to inducecytotoxicity. In these embodiments, the controller 42 is programmed tocycle the control input through the states that cause the AC voltagegenerator 44 to output the second frequency and the one or moreadditional frequencies before the request arrives. The controller 42 isalso programmed to accept a request to switch to the first frequency.Upon receipt of the request, the controller 42 will place the controlinput in the first state so that the AC voltage generator 44 will outputthe first frequency for an interval of time (e.g., 72 hours). After theinterval of time has elapsed, the controller 42 will revert to cyclingthe control input through the states that cause the AC voltage generator44 to output the second frequency and the one or more additionalfrequencies.

The system depicted in FIG. 10 is particularly useful when a person hasa tumor that is being treated by combination therapy that includesTTFields and chemotherapy. In this situation, the system operates mostof the time at the second frequency to provide the maximum cytotoxicityeffect. But before a person visits a chemotherapy clinic for a dose ofchemotherapy, healthcare personnel (or the user) actuates the userinterface 40 to switch the system to the first frequency that promotesBBB permeability. In this situation, the actuation of the user interfacecould be done e.g., 72 hours before the expected start of thechemotherapy.

Alternatively, upon receipt of the request (e.g., from the userinterface 40), the controller 42 can control the control input so thatthe AC voltage generator 44 will output the first frequency for aninterval of time (e.g., 1 hour), then switch back and forth between thesecond frequency and the first frequency (e.g., switching every hour).Eventually (e.g., when the relevant substance has been exhausted fromthe patient's bloodstream), the controller 42 controls the control inputso that the AC voltage generator 44 reverts to outputting the secondfrequency.

A set of electrodes (not shown) that are similar to the conventionalelectrodes used with Optune® are connected to the output of the ACvoltage generator 44.

Note that in connection with any of the methods described above, the BBBshould recover to its original low-permeability state after a sufficientamount of time has elapsed following the termination of the alternatingelectric field. This can be important in many contexts for the safety ofthe subject.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the sphere andscope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

What is claimed is:
 1. A method for treating a tumor in a subject'sbrain and delivering a substance across a blood brain barrier of thesubject's brain, the method comprising: applying a first alternatingelectric field at a first frequency to the subject's brain for a firstperiod of time, wherein application of the first alternating electricfield at the first frequency to the subject's brain for the first periodof time increases permeability of the blood brain barrier in thesubject's brain; administering the substance to the subject after thefirst period of time has elapsed, wherein the increased permeability ofthe blood brain barrier allows the substance to cross the blood brainbarrier; and applying a second alternating electric field at a secondfrequency to the subject's brain for a second period of time that is atleast one week long, wherein the second frequency is different from thefirst frequency, wherein the second alternating electric field at thesecond frequency has an intensity that is sufficiently large to inhibitthe tumor.
 2. The method of claim 1, wherein the first frequency isbetween 75 kHz and 125 kHz.
 3. The method of claim 1, wherein the firstfrequency is between 50 kHz and 190 kHz.
 4. The method of claim 3,wherein the second frequency is between 190 kHz and 210 kHz.
 5. Themethod of claim 1, wherein the first period of time is at least 24hours.
 6. The method of claim 1, wherein the second period of timecomprises a single uninterrupted interval of time that is at least oneweek long.
 7. The method of claim 1, wherein the second period of timecomprises a plurality of non-contiguous intervals of time during whichthe second alternating electric field at the second frequency is appliedto the subject's brain, wherein the plurality of non-contiguousintervals of time collectively add up to at least one week.
 8. Themethod of claim 1, wherein the substance has a molecular weight of atleast 4 kDa.
 9. The method of claim 1, wherein the substance has amolecular weight of at least 69 kDa.
 10. The method of claim 1, whereinthe substance has at least one characteristic that ordinarily impedesthe substance from crossing a non-leaky BBB.
 11. An apparatus fortreating a tumor in a subject's body and facilitating delivery of asubstance across a blood brain barrier of the subject's body, theapparatus comprising: an AC voltage generator capable of operating at afirst frequency between 50 and 190 kHz and a second frequency between 50and 500 kHz, wherein the second frequency is different from the firstfrequency, the AC voltage generator having a control input, wherein theAC voltage generator is configured to output the first frequency whenthe control input is in a first state and to output the second frequencywhen the control input is in a second state; and a controller programmedto (a) place the control input in the second state so that the ACvoltage generator outputs the second frequency, (b) accept a request toswitch to the first frequency, (c) upon receipt of the request, placethe control input in the first state so that the AC voltage generatoroutputs the first frequency for an interval of time, and (d) after theinterval of time has elapsed, place the control input in the secondstate so that the AC voltage generator outputs the second frequency. 12.The apparatus of claim 11, further comprising: a set of electrodesconfigured for affixation to the subject's body; and wiring thatconnects an output of the AC voltage generator to the set of electrodes.13. The apparatus of claim 11, wherein the first frequency is between 75kHz and 125 kHz, and the second frequency is between 150 kHz and 250kHz.
 14. The apparatus of claim 11, wherein the interval of time is atleast 24 hours.
 15. The apparatus of claim 11, wherein the interval oftime is at least 72 hours.
 16. The apparatus of claim 11, wherein thecontroller is further programmed to, subsequent to the receipt of therequest, switch the control input back and forth between the first stateand the second state.
 17. The apparatus of claim 11, wherein the ACvoltage generator is capable of operating at at least one additionalfrequency between 50 and 500 kHz, and wherein the AC voltage generatoris configured to output the at least one additional frequency when thecontrol input is in at least one additional state, and wherein thecontroller is programmed to cycle the control input through the secondstate and the at least one additional state prior to receipt of therequest, and to cycle the control input through the second state and theat least one additional state after the interval of time has elapsed.18. The apparatus of claim 11, further comprising a user interface,wherein the request is accepted via the user interface.
 19. Theapparatus of claim 11, wherein the request is accepted via RF.